Factor VIII is a plasma glycoprotein involved in blood coagulation. Deficiency or abnormality in its function results in severe hereditary disease called hemophilia A (Eaton, D. et al., 1986, Biochemistry 25: 505-512; Toole, J. J. et al., 1984, Nature 312: 342-347; Vehar, G. A. et al., 1984, Nature 312: 337-342). Up to now, the only treatment for hemophilia A has been intravenous administration of factor VIII prepared from human blood or a recombinant source. Due to the safety reason, recombinant factor VIII has been preferred to plasma derived factor VIII. However, since expression level of factor VIII is 2-3 order magnitudes lower than other molecules in the same expression system (Lynch C. M., 1993, Human Gene Therapy 4: 259-272), recombinant factor VIII production has not met its demand.
Several attempts have achieved an improved expression of factor VIII by removing B-domain which has been known not to have any function in the cofactor activity of factor VIII (Eaton et al., 1986, Biochemistry 25:8343-8347; Burke, R. L. et al., 1986, J. Biol. Chem., 261: 12574-12578; Toole, J. J. et al., 1986, Proc. Natl. Acad. Sci. USA, 83: 5939-5942; Fay et al., 1986, Biochem. Biophys. Acta, 871:268-278). U.S. Pat. No. 5,661,008 and WO-A-91/09122 described B-domain deleted versions of factor VIII, which is similar to the shortest form of plasma factor VIII. U.S. Pat. Nos. 5,112,950 and 7,041,635 disclosed the single chain forms of B-domain deleted factor VIII molecules.
As a choice for the mammalian cell expression, Chinese Hamster Ovary (CHO) cell expression system has been used in producing many therapeutic proteins including factor VIII (Chu, L et al., 2001, Curr. Opin. Biotehnol., 12: 180-187). The characteristics of CHO cell line are elucidated. It can grow either in anchorage dependent manner or in suspension manner, adapt to either serum-containing medium or serum-free medium, and especially support post-translational modifications of proteins nearly identical to the human glycosylation patterns (Brooks S. A., 2004, Mol. Biotechnol., 28: 241-255; Jenkins, N., et al., 1996, Nat. Biotechnol., 14: 975-981; Chen, Z., et al., 2000, Biotechnol. Lett., 22: 837-941; Mols, J., et al., 2005, 41: 83-91). CHO cell lines producing therapeutic proteins have been usually cultured in the animal-derived protein free medium for the purpose of addressing safety concerns about transmission of animal derived virus or prion and for the purpose of easier purification. (Chu, L., et al., 2001, Curr. Opin. Biotehnol., 12: 180-187). However, removal of serum from the cultivating media also deprives the naturally contained protease inhibitors in a serum supplement and makes it difficult to maintain the viability of the cells during the production processes (Mols, J., et al., 2005, 41: 83-91; Sandberg, H., et al., 2006, Biotechnol Bioeng., 95: 961-971).
Reduced viability and stressful conditions seem to increase the production of secreted or released proteases from dead cells which can attack the therapeutic proteins and cause heterogeneity. Heterogeneity caused by internal cleavages of therapeutic protein might be the major problem because cleaved proteins can be inactive and make it difficult to maintain “lot to lot” consistency during the production and purification processes. Therefore, it is important to maintain a relatively low level of protease or to prevent protease activity during production.
A few successful efforts to prevent this proteolysis caused by released proteases from CHO cell line during culture have been reported, even though universal inhibitor(s) which could apply to all therapeutic proteins produced in CHO cell line has not yet been found. Satoh M et al. reported the presence of cystein and serine proteases released from CHO cell. Chotteau et al. (Chotteau, V., et al., 2001, in Animal cell technology: from target to market, Kluwer Academic publishers, pp. 287-292) found that an unidentified, extracellular metal-dependent protease from CHO cell culture medium was responsible for the proteolysis of truncated factor VIM In WO-A-90/02175, it is disclosed that some serine or cysteine proteases from CHO cell culture can be blocked by the inhibitor peptides, which increase factor VIII productivity. EP A 0 306 968 discloses addition of aprotinin to culture medium increased expression of factor VIII in CHO cell medium by three times.
In U.S. Pat. No. 5,851,800, inventors claimed the inhibitors of metalloproteases and chymotrypsins could reduce detrimental effect on factor VIII production in cell culture. Sandberg H. et al. characterized two types of proteolytic activities released by CHO cells in a cell culture. One was originated from metalloproteinases, and the other from serine protease. Only metalloproteinases was found to have a strongly negative effect on the factor VIII activity. However, even though inhibitor of metalloproteases such as EDTA and 1,10 o-phenantroline could block the factor VIII cleavage as described by Sandberg H. et al., these inhibitors cannot be directly added into the CHO cell culture medium due to its toxic effect on cells, judged from our experiments.
All the above-mentioned protease inhibitors and commercially available protease cocktail which contain inhibitors against serine, cystein, aspartic and aminopeptidases such as aprotinin, bestatin, leupeptin, E-64 and pepstatin A have been applied to our single chain factor VIII derivative (described in U.S. Pat. No. 7,041,635) culture, but we found that none of them were effective in protecting our factor VIII derivative from cleavage by released protease(s) from CHO cell culture during the culture.
U.S. Pat. No. 6,300,100 discloses sulfated polysaccharide such as heparin protected an intact Tissue Factor Pathway Inhibitor (TFPI) from cleavage by proteases present in the culture medium. In addition, U.S. Pat. No. 5,112,950 discloses sulfated dextran to substitute the stabilizing effect of Von Willebrand factor on factor VIII in serum free media. However, to our knowledge, there has been no report on the inhibitory effect of dextran sulfate against proteases in connection with factor VIII molecules.
The present invention aims to demonstrate the protective effect of dextran sulfate on the cleavage of Factor VIII or its derivatives from proteases produced during CHO cell culture.